(A) Experimental Laboratory Component: Using reverse genetics, low pathogenic avian influenza (LPAI) viruses (H5N1 and H6N1) with the minimal multibasic cleavage site (MBCS) motif will be generated and the intravenous pathogenicity index (IVPI) and 50% bird infectious dose (BID50) will be determined in chicken and compared to LPAI H5N1 and H6N1. Next, these viruses will be serially passaged (10x) in chicken to see whether the minimal MBCS will evolve into a MBCS and the highly pathogenic avian influenza (HPAI) phenotype. Deep sequencing (RML Research Technologies Section, Genomics Unit) will be used to analyze the cleavage site motif of viruses from chicken in each passage. (I) Generation of recombinant viruses: We have started with cloning efforts to establish the reverse genetics systems for LPAI H5N1 and H6N1. This work is currently on hold due to the H5 moratorium. (II) Determination of IVPI and BID50 of recombinant viruses: This work is currently on hold due to the H5 moratorium. (III) Passage of recombinant viruses in chicken: This work is currently on hold due to the H5 moratorium. (IV) Sequence analysis of cleavage site: This work is currently on hold due to the H5 moratorium. (B) Field Survey Component: Historically, the natural reservoir for all influenza A viruses were considered wild birds. However, the recent identification of a novel H17 influenza A virus in yellow-shouldered bats in Guatemala has put the focus on novel reservoirs besides wild birds. Certain influenza A viruses in particular are a direct threat to both animal and public health. In particular, Highly Pathogenic Avian influenza A viruses (HPAI H5 and H7 subtypes) and a wide variety of swine influenza viruses (H1, H2 and H3) are notorious for crossing the species barrier from animals to humans. Public health, animal health, agricultural productivity and food security in the Republic of Congo (RC) and the wider Congo basin region are directly threatened by outbreaks of influenza A viruses. In addition, the potential zoonotic transmission of influenza viruses to the human population could spark outbreaks and the emergence of a new pandemic. The recent zoonotic influenza A virus transmissions in various African countries (i.e. Nigeria, Egypt, Niger, Cameroon, Sudan, Burkina Faso, Djibouti and Ivory Coast) highlight the potential for influenza A viruses to evolve into global veterinary and public health threats. To date little or no information has been published on the prevalence, incidence and identity of animal influenza viruses, in particular avian influenza viruses, in RC and there are currently no surveillance programmes for avian influenza viruses in place. Therefore, there is a real need to develop baseline information with reference to the circulation of avian influenza viruses. (I) To obtain contemporary information on the circulation of avian influenza A viruses in wild and domestic avian and mammal populations in RC and neighboring countries. (II) To identify and characterize isolated avian influenza A viruses by molecular diagnostic tools. (III) To use the data generated in the project to carry out science-based risk assessments and to develop appropriate intervention strategies in order to prevent and control the spread of avian influenza A viruses. This project has just very recently been implemented and surveillance efforts have started.